Chromosome segregation is one of the most critical processes common to every organism. To ensure proper segregation, the cell employs a checkpoint mechanism to monitor defects in the segregation machinery. The mitotic checkpoint ensures that even a single improperly attached chromosome is capable of delaying the metaphase to anaphase transition. To achieve a delay prior to chromosome segregation, the checkpoint inhibits, through a poorly understood mechanism, the anaphase-promoting complex/cyclosome (APC/C). Our preliminary studies revealed that injection of antibodies to the c-terminus of Cdc27, a subunit of the APC/C, abrogated the mitotic checkpoint in HeLa cells. This is supported by preliminary studies in yeast which revealed a c-terminal deletion of Cdc27 that prematurely separates its sister chromatids in the presence of nocodazole. To extend these observations, I propose to use both genetic and biochemical approaches in the budding yeast Saccharomyces cerevisiae and in a mammalian tissue culture system to characterize the interaction between Cdc27 and the mitotic checkpoint and test how this interaction might facilitate mitotic checkpoint inhibition of the APC/C.